PROJECT SUMMARY Transient Receptor Potential (TRP) channels are regulated by a broad range of stimuli, including chemicals, temperature, mechanical stress and membrane voltage and are directly involved in the perception of sensory modalities such as vision, taste, olfaction, hearing, touch, temperature and pain. TRP channels are implicated in the pathogenesis of numerous diseases and some of them represent important prognostic markers and promising targets for new therapeutic strategies to treat a variety of human cancers. For efficient drug design, we need detailed information about TRP channel structure and function. We plan to study TRP channel structure and function using a combination of different biophysical and biochemical methods. Our specific aims are: 1) establish molecular bases of TRP channel regulation by calcium, 2) determine the molecular mechanism of TRP channel gating, and 3) develop molecular models of TRP cannel inhibition by various organic and inorganic molecules. TRP channels are challenging targets for structure-functional studies because they represent multimeric integral membrane proteins of a large size with typically low expression levels. To achieve our goals, we will use a combination of structural and functional approaches including modern crystallographic techniques, Fluorescence-based Size Exclusion Chromatography (FSEC), calcium imaging, fluorescent spectroscopy and electrophysiology. We will use different crystallization methods and temperatures, screen detergents, lipids and ligands to obtain structures of intact TRP channels in different conformational states. We will then combine the nascent structural information with functional data to discern mechanisms of TRP channel regulation and gating. Achieving our aims will have a significant impact on sensory biology and will result in a new structural/functional model of TRP channel that can serve as a dynamic template for theoretical prediction, in silico fitting and chemical synthesis of new drugs.